生物信息学论文

.2007,3

471

InternationalJournalofBiologicalSciences

20073(7):471-476

©htsrerved

RearchPaper

TheGrowth-hormoneinducibletransmembraneprotein(Ghitm)belongsto

theBaxinhibitoryprotein-likefamily

KerstinReimers*,ClaudiaYUChoi*,VesnaBucan,PeterMVogt

DepartmentforPlastic,HandandReconstructiveSurgery,MedicalSchoolHannover,Podbielskistraße380,D-30659

Hannover,Germany

*Theauthorscontributedequally.

Correspondenceto:KerstinReimers,Ph.D.,KlinikfürPlastische,Hand-undWiederherstellungschirurgie,MedizinischeHochschule

Hannover,Podbielskistraße380,:+49-511-9063917;Fax:+49-511-9063480;E-mail:

n@

Received:2007.07.23;Accepted:2007.11.20;Published:2007.11.22

TheconrvedproteindomainUPF0005isaproteinfamilysignaturedistributedamongmanyspeciesincluding

ghofunknownfunctionalitythismotifhasbeenfoundinnewlyidentified

antiapoptoticproteinscomprisingtheBI-1family,namelyBax-inhibitoryProtein-1(BI-1),Lifeguard(LFG),and

rchforvertebrateproteinspresumablybelongingtotheBI-1family,wefoundthat

Growth-hormoneinducibletransmembraneprotein(Ghitm)isanotherprospectivememberoftheBI-1family.

HerewecharacteriGhitminafirstanalysisregardingitsphylogeny,expressionincancercelllines,and

proteomicalproperties.

Keywords:proteinfamily,Bax-inhibitor,programmedcelldeath,bioinformatics,apoptosisinhibitors,transmembrane

protein

uction

TheevolutionarilyconrvedmotifUPF0005,

althoughfunctionallyuncharacteridsofar,isfound

inaproteinfamilywildlydistributedamongall

oteinsbearingthismotifhavebeen

identifiedandincorporatedintothepublicdatabas

includingthreenovelantiapoptoticproteins,Bax

Inhibitor-1(BI-1),Lifeguard(LFG),

beenpropodthatBI-1andLFGandprobablyother

clolyrelatedproteinsreprentanewfamilyof

cytoprotectiveproteinscalledBI-1family[1].

DuetothehydrophobicnatureoftheUPF0005

motif,ithasbeenpredictedthatitinvolvessixtoeight

mental

evidenceaswellasbioinformaticanalysisindicatethat

proteinsbelongingtothisfamilymightresideinthe

membraneoftheendoplasmaticreticulumandare

involvedintheregulationofcelldeathcontrolbythe

Bcl-2family[2-4].

BI-1isthefarmostbestcharacteridmemberof

iapoptoticfunctionofBI-1was

detectedbyascreenforfunctionalrepressorsofthe

proapoptoticpropertiesofBaxinyeast[4].LFGhas

beenshowntoprotectagainstFas-inducedapoptosis

inmammaliancells[5].H-GAAPisaninhibitor

againstintrinsicandextrinsicstimuliwithahighly

conrvedcounterpartfoundinvacciniavirus[6].

BI-1isup-regulatedinveralmalignanttumors

includingpulmonaryadenocarcinoma,breastcancer,

lymphomaandprostatecancer,whilstGhitmwas

foundtobeover-expresdinbreastcancers[7-10].

ForabettercharacterisationoftheBI-1family,we

archedthedatabaforproteinscontainingthe

asizedthatthe

proteinsshouldhavehomologuesinhighervertebrate

speciesandexpressiondatamightindicatea

functionalroleintheregulationofcrucialcellular

heproteinsfoundwastheGrowth

hormone-inducibletransmembraneprotein(Ghitm).

Ghitmwasfirstidentifiedinthebrownadipotissue

frommou[9].Ghitmisubiquitouslyexpresdin

mammaliancellswithrelativelylowexpressionin

intestineandthymus[11].Itwaspropodthatit

functionsintumorigenesisandinadipotissues[9],

althoughafunctionalmechanismhasnotbeen

described.

WeanalydtherelationbetweenLFG,BI-1,and

Ghitminaphylogeneticanalysisandud

bioinformaticaltoolstosummarithemolecular

essionanalysisfor

ghitmtranscriptswasperformedforsomecancercell

lines.

alandMethods

Phylogenetictreeconstruction

AminoacidquencesudinCLUSTALW

analysisandphylogeneticanalysiswiththeiraccession

numbersaregiveninbrackets:sF40F9

(CAA94766),chickenLFG(XP_424507),humanLFG

(NP_036438),mouLFG(NP_082500),ratLFG

(NP_653357),XenopusBI-1(AAH47131),humanBI-1

.2007,3

472

(AAH36203),ratBI-1(P55062),mouBI-1

(AAH05588),macaqueBI-1(AAV98554),Xenopus

Ghitm(NP_001017195),XenopusGhitm-prov

(AAH41226),humanGhitm(CAH72661),human

Ghitm(CAG38550),ratGhitm(NP_001005908),mou

Ghitm(NP_510963),dogGhitm(XP_536408),cow

Ghtim(NP_001029224),zebrafishGhitm(NP_956885),

chickenGhitm(NP_001026388),dogGAAP

(XP_531662),humanGAAP(AAF14868),mou

glutamatereceptor(NP_075657),putativeMAPK

activatingprotein(BAC77379),humanRECS1

(Q969X1).SequenceswerealignedusingClustalW.

ThesoftwarepackagePHYLIP3.64wasudfor

tdistprogramwith

Jones-Taylor-Thorntonmatrixwasudtogeneratea

ghbor-joiningalgorithm[12]

andthemaximumlikelihoodmethodintergratedin

thesoftwaretoolsNeighbor-joiningandPROTML,

respectivelywereudforgenerationofphylogenetic

tandConnprogramswereudto

statisticallyasssthestrengthofthetreesusing

nsustreewasviewed

withTreeView(/

rod/).ClustalWwasudtodetermine

thepercentageofquenceidentitybetweentheamino

acidquencesofGhitmfromdifferentvertebrate

species.

Sequenceanalysis

NCBIRPS-BLASTwasudtoarchfor

conrveddomaindatabascreening

(/structure/cdd/wrpsb

.cgi).

Sequenceanalysiswasperformedwiththe

primaryaminoacidquenceofhumanandmou

ndDoolittlehydropathicplot[13]was

generatedwithProtScale(/

cgi-bin/)withKyteandDoolittleoption.

Proteintopologywaspredictedwith[14,15]

TOPPRED2(/qanal/inter

faces/),TMPRED(

/software/TMPRED_),TMHMM

(/rvices/TMHMM/)

HMMTOP(/hmmtop/html/

adv_),andSMART(-

).

SignalPwasudforthepredictionofsignal

peptidesandthepossiblelocalisationwascalculated

withPSORTII[16]:/SignalP:

(/rvices/SignalP/)TargetP:

/rvices/TargetP/).

Sequencepatternandmotifarchwasperformed

withthevarioustoolscollectedattheEXPASY

proteomicsrver(/).

Geneexpressionanalysis

TotalRNAwasisolatedfromthecancercelllines

andrevertranscribedinareactioncontaining0.25

mMdNTP-mix,1µgrandomhexamers,20U

recombinantStratascriptII(Stratagene)with1x

Stratascriptbuffersuppliedbythemanufacturer

followinginstructions.1µlofthereactionmixturewas

udinpolymerachainreactionwiththespecific

primers(n5’-GGGCCTGGGTCTCGTCTT

TG-3’;antin5’-ATCCACCGTACATTGCCA

CTGAG-3’).Cyclingconditionswere35cyclesof94°C

for30s,65°Cfor30sand72°

amplificationproductswereanalydona2%agaro

gelsupplementedwithEtBr.

sandDiscussion

GhitmisanewmemberoftheBI-1family

WeidentifiedmembersoftheBI-1familyby

allotherfamilymembersGhitmhastheUPF0005

acteri

therelationbetweenGhitm,BI-1andothermembersof

theBI-1familywelectedquencesofdifferent

vertebratespeciesandarrangedtheminmultiple

nClustalWalignment

phylogenetictreeswereconstructedasdescribedin

aare

prentedinaphylogenetictreeusingtheUPF0005

motifcontaininghypotheticalproteinF40F9fromC.

Elegansasanoutgroup(figure1).Ghitmisfoundina

cladetogetherwithBI-1,whiletherecentlydescribed

apoptosisinhibitoryproteinh-GAAPismoredistantly

rgroupisbuildbytheantiapoptotic

proteinLFGandthehumansofaruncharacterid

putativeMAPKactivatingproteinandtherelated

murineN-methylD-asparate-associatedprotein1.

Allspeciesarefoundinaccordancewiththe

genealogicaltree.

HomologuesofGhitmhavebeenfoundinmany

differentvertebratespeciesasdepictedinfigure1.

Sequenceidentityvaluesforthevertebrate

homologuesrangebetween60.3%(zebrafishandrat)

and93.4%(dogandhuman).Forhumanandthe

clawedfrogtwonearlyidenticalquenceshavebeen

found(human:93.2%;clawedfrog:93.1%).Additional

analysisisnecessarytocommentonthenatureofthe

similaritiesanddifferencesbetweenthequences.

Ahighdegreeofquenceconrvationamong

differentspeciesisatypicalfeatureforthemembersof

thatBI-1

homologuesfromBrassicanapusandNicotiana

tabacumsuppressBax-inducedapoptosisinhuman

-1inhibitsmammalianBax-inducedcell

deathinyeastcells,andplantsimplyingfunctional

conrvationinanancientmechanismof

cytoprotection[4,17].ForLFGandh-GAAP

homologueshavebeenidentifiedformanydifferent

species[1,6].ForGhitm–apartfromthevertebrate

quencesdescribedinthisstudy–quencesfor

non-vertebratespecieshavealsobeenfoundsuchas

fruitflyandnematode[11].

.2007,3

473

Figure1:PhylogenetictreeoflectedmembersoftheBI-1familybadonClustalWalignmentofthededucedaminoacid

medproteinwith

s(CAA94766F4)rapvaluesaregivenateachnode.

BioinformaticalcharacterisationofGhitm

KyteandDoolittlehydrophobicityplotshave

beencarriedoutfortheaminoacidquencesof

human,sahydrophobic

patternfortheexaminedspecieshuman,mouand

clawedfrogwith6dominantspikes(figure2a-c).

AccordingtotheKyteandDoolittlepatternwe

assumedthatGhitmisapolytopicmembraneprotein

with6transmembrane(TM)domains(figure2d).The

propodtopologyhadbeenarrangedwiththeaidof

heirpredictions

Ghitmbelongstothemembraneproteinstype3aand

probablyresideitherintheendoplasmaticreticulum

(ER)orintheplasmamembraneaccordingtoPSORT

II,llularlocalisationin

theendoplasmaticreticulumhasbeenassumedfor

BI-1andLFGaswell[1-3].Incontrasttothepossible

proteintopologyofLFGitwaspredictedforGhitm

thattheN-terminusaswellastheC-terminusare

foundonthecytoplasmaticsideoftheERmembrane,

whiletheshortN-terminusofLFGisprobablyfound

intheERlumen.

Interestingly,ithasbeenreportedforGhitmthat

itposssanN-terminalsignalpeptideandthat

N-terminalcleavageisimportantfortheregulationof

fullylookedatthe

N-terminioflectedquencesofvertebrateGhitm.

ThesignalpeptidepredictionprogramSignalPusa

combinedneuralnetworkandhiddenMarkovmodels

forthepredictionofsignalpeptidesandtheircleavage

aareexpresdinscorevaluexpressing

theoutputfromthesignalpeptide/nonsignalpeptide

networks(S-score),theoutputfromthecleavage

site/non-cleavagesitenetworks(C-score),and

geometricaverageoftheC-scoreandasmoothed

derivativeoftheS-score,termedY-score[18].The

N-terminalquencesoflectedvertebrateGhitm

ologues

ofGhitmfoundinthelowervertebratesarepredicted

tohaveasignalpeptide,whiletheprobability

extremely

interesting,takingintoconcernthatYoshidaetal.

foundoutthatcleavageoftheN-terminalsignal

peptideisntialforproteinexpressionandfunction

inthemou[11].Acriticalexaminationofthe

functionofthesignalpeptideduringtheevolutionof

theGhitmproteinisneeded.

Infigure4anoverviewovertheproteinstructure

offourknownmembersoftheBI-1familyisgiven

withtheputativetransmembranedomainsunderlined.

Itisclearlyvisiblethat,whileallfourproteinsare

highlysimilar,thetwoven-spantransmembrane

proteinsLFGandRECS1areorganizedthesamewith

aprolineandglycinerichintracellularN-terminus

.2007,3

474

containingveralPXXPmotifsandonepotential

PESTquence[19].Thetwosix-spantransmembrane

proteinsBI-1andGhitmdiffermoreintheir

arrangementofputativetransmembranedomains.

N-terminusandC-terminusarepredictedtobeinside

thecellandbotharecharacterizedbyanumberof

basicresidues:arginine10%frequency,lysine8.75%

frequencyattheN-terminus,arginine5.66%

frequency,lysine9.34%frequencyattheC-terminus.

IncontrasttoRECS1whichhasaprolinecontentof

24%inthehumanquenceprolineresiduesarefound

toamuchlesrextentinGhitm(N-terminus:6.25%,

C-terminus3.77%).PXXPandPPXYmotifshavenot

beenfound.

Ghitmexpressionincancercelllines

Ghitmgeneexpressioninveralcancercelllines

icprimerswere

correspondingtotheexpectedsizearedetectedinthe

cDNAsderivedfromallcancercelllinestestedthatis

A549lungcarcinomacells,ACTLlymphomacells,

HeLacervixcarcinoma,HepG2hepatoma,SW872

liposarcoma,MCF-7breastcancer,SW-620colon

carcinoma,andU2-OSosteosarcomacells(figure5).

AdirectcytoprotectivefunctionofGhitmhasnot

beendemonstratedsofar,butobrvationspublished

intheliteraturesuggestaroleinenergymetabolism,

and,withregardtoitsassociationtothegrowth

hormonesignalling,inagingandimmunology[9,11].

Thefunctionsmightbelinkedtoacytoprotective

roleinthecellularaccomplishmentofERstressbut

mustbeevaluatedincorrespondingexperimental

designs.

sion

AlthoughtheprecifunctionoftheC-terminal

domainUPF0005oftheBI-1familyisnotknown,a

biologicalfunctionissuggestedbythehighdegreeof

quenceconrvationbetweenspecies.

ComputationalanalysisrevealedthatGhitmsharesthe

UPF0005motifwiththecytoprotectivemembersofthe

BI-1familymembers,andisclolyrelatedtoBI-1,

h-GAAP,ghthereisstillaneedfor

moreexperimentalevidencetheproteinshavemany

dicted

proteintopologyindicatesthattheyare

transmembraneproteinsofthetype3awithavariable

numberoftransmembranespannersfromsixtoeight

lity

wasfoundintheN-terminiprobablydueto

modificationsinbiologicalmechanismswhichhave

rstudiesareneededto

elucidatethephysiologicalandprobablypathological

functionofGhitm.

Figure2:Proteintopologypredictionbadonthe

d

Doolittleplotsofthededucedaminoacidquencesofhuman

(a),mou(b),andclawedfrog(c).TheputativeTMgments

arevisibleashydrophobespikes.(d)Calculatedtransmembrane

domainorganisationofGhitmwithsixmembranepassagesand

cytoplasmicN-andC-terminalends.

.2007,3

475

Figure3:GhitmN-siblecleavagesiteas

comeoftheprobabilityscorecalculatedwithSignalPisgivenatthe

right.

Figure4:DeducedaminoacidquencesofthehumanBI-1,LFG,RECS1,calaminoacids

areillustratedbystars;ativetransmembranedomainscalculatedbyTMHMMare

indicatedbyverticalbarsbelowthequences.

.2007,3

476

Figure5:GeneexpressionofGhitmisfoundinveralcancer

celllines(a).18SrRNAamplificationisudasareaction

control(b).1notemplatecontrol,2A549,3ACTL,4HeLa,5

HepG2,6MCF-7,7SW-620,8SW-872,9U2-OS

Acknowledgement

Theworkwasfundedbythe

authorsaregratefultoChristinaAllmelingforhelpful

commentsonmanuscriptandfiguresandthank

AndreaLazaridisforhelpwiththegeneexpression

analysis.

Conflictofinterest

Theauthorshavedeclaredthatnoconflictof

interestexists.

References

sK,ChoiCY,Mau-ThekE,ceanalysisshows

thatLifeguardbelongstoanewevolutionarilyconrved

lMed.2006;18:729-34.

,KeN,KimHR,ionarilyconrved

cytoprotectionprovidedbyBaxInhibitor-1homologsfrom

animals,plants,.2003;323:101-13.

-OhoriY,NaganoM,MutoS,athsuppressor

Arabidopsisbaxinhibitor-1isassociatedwithcalmodulin

hysiol.2007;143:650-60.

ibitor-1,amammalianapoptosis

l.

1998;1:337-46.

V,SchmittMJ,VetterDE,:ananti-apoptotic

NatlAcadSciUSA.1999;96:12667-72.

C,BergamaschiD,HollinsheadM,hibitor

thog.

2007;3:e17.

M,ThelenP,HemmerleinB,ibitor-1is

overexpresdinprostatecanceranditsspecific

down-regulationbyRNAinterferenceleadstocelldeathin

hol.2003;163:543-52.

M,KaulfussS,ThelenP,sionandfunctional

l.

2006;208:340-9.

,KelderB,fication,isolation,andcloning

ofgrowthhormone(GH)-inducibleinterscapularbrownadipo

complementarydeoxyribonucleicacidfromGHantagonistmice.

Endocrinology.2001;142:2937-45.

R,IshiyamaT,UchiharaT,sionoftheBax

.2006;

106:648-53.

aT,NagataS,sanorthologofthe

Bombyxmoriprothoracicgland-derivedreceptor(Pgdr)thatis

ubiquitouslyexpresdinmammaliancellsandrequiresan

mBiophysRes

Commun.2006;341:13-8.

ghbor-joiningmethod:anewmethodfor

lEvol.1987;4:406-25.

emethodfordisplayingthe

ol.1982;157:105-32.

ilityoftransmembrane

tt.2002;532:415-8.

,KroghA,ilitymeasuresfor

ol.

2003;327:735-44.

:aprogramfordetectingsorting

signalsinproteinsandpredictingtheirsubcellularlocalization.

TrendsBiochemSci.1999;24:34-6.

N,OuelletM,PitreF,larcharacterizationof

twoplantBI-1homologueswhichsuppressBax-induced

.2003;216:377-86.

nH,EngelbrechtJ,BrunakS,ficationof

prokaryoticandeukaryoticsignalpeptidesandpredictionof

nEng.1997;10:1-6.

,ItoA,KimuraSH,1deficiencyinmice

GenetSyst.2006;81:41-50.